The covalent attachment of polymers to therapeutic proteins (such as the addition of poly(ethyleneglycol) (PEG) to interferon) has led to extensive commercial use. Since the advent of protein PEGylation in 1979 (Veronese, F. M. Biomaterials 2001, 22, 405), much of the research devoted to improving the efficacy of protein therapeutics has been focused on increasing circulation time and reducing immunogenicity. As of 2011, at least nine PEGylated protein drugs had been approved by the U.S. Food and Drug Administration (FDA) for treatment of diseases including Hepatitis C (O'Sullivan, A. K.; Buti, M.; Delong, K.; Prasad, M.; Sabater, F. J.; Esteban, R.; Weinstein, M. C. Value Health 2008, 11, A437), acute lymphoblastic leukemia (Dinndorf, P. A.; Gootenberg, J.; Cohen, M. H.; Keegan, P.; Pazdur, R. Oncologist 2007, 12, 991.), and Crohn's disease (Nesbitt, A.; Fossati, G.; Bergin, M.; Stephens, P.; Stephens, S.; Foulkes, R.; Brown, D.; Robinson, M.; Bourne, T. Inflamm Bowel Dis 2007, 13, 1323; Alconcel, S. N. S.; Baas, A. S.; Maynard, H. D. Polym Chem-Uk 2011, 2, 1442.). While PEGylation is a useful tool to hide protein based therapeutics from the immune system, and to increase size to slow elimination from the body, little additional specific functionality is added by PEGylation. In recent years, targeted drug delivery and drug carriers with responsive functionality (Chilkoti, A.; Dreher, M. R.; Meyer, D. E.; Raucher, D. Adv Drug Deliver Rev 2002, 54, 613; Su, J.; Chen, F.; Cryns, V. L.; Messersmith, P. B. J Am Chem Soc 2011, 133, 11850; Nasongkla, N.; Bey, E.; Ren, J.; Ai, H.; Khemtong, C.; Guthi, J. S.; Chin, S.-F.; Sherry, A. D.; Boothman, D. A.; Gao, J. Nano Lett 2006, 6, 2427) have been investigated to improve efficacy of current therapeutics.
Polymer conjugation to proteins can be completed using one of two methods: “grafting to” or “grafting from.” In “grafting to,” pre-synthesized, end functionalized polymers are coupled to accessible amino acid side chains or end termini on the protein surface. The “grafting-to” technique dominates the literature. The grafting site of a functionalized synthetic polymer to a protein surface through a coupling reaction is often a random process in which the density and site(s) of the grafted polymer cannot be controlled. Naturally, once a first polymer chain has “grafting-to” the protein surface steric hindrance will often prohibit further polymer binding to near-by sites on the protein surface, resulting in a low density of the grafting polymer. (Lele, B. S.; Murata, H.; Matyjaszewski, K.; Russell, A. J. Biomacromolecules 2005, 6, 3380-3387; Yang, Z.; Domach, M.; Auger, R.; Yang, F. X.; Russell, A. J. Enzyme Microb. Technol. 1996, 18, 82-89.) Although “Grafting to” techniques provide a wide range of polymerization reactions and monomers to select from, a large excess of polymer is often required to overcome steric limitations caused by coupled polymers. In addition, separation of protein-polymer conjugates from unreacted polymer can prove to be difficult when using the “grafting to” method.
Many pH-responsive polymers show a reversible phase transition between expanded and collapsed forms due to ionization and deionization of the side groups on the polymer that leads to alteration in hydrodynamic volume and solubility in aqueous media. Early studies on pH-responsive polymer-protein conjugates showed that conjugation of carboxylated polymers such as poly(acrylic acid) to proteins influenced the pH dependence of solubility and activity. (see Charles, M.; Coughlin, R. W.; Hasselberger, F. X. Biotech. Bioeng. 1974, 16, 1553-1556; Van Leemputten, E.; Horisberger, M. Biotech. Bioeng. 1976, 18, 587-590) Thermo-responsive polymers respond to changes in temperature and exhibit reversible transitions between collapsed and expanded forms at temperatures above and below their critical solution temperature. For example, poly(N-isopropylacrylamide) (pNIPAm) has a low critical solution temperature (LCST) around 32° C. in the aqueous solution. At temperatures above the LCST, pNIPAm becomes dehydrated and collapses into micelle-like particles which precipitate from solution. This property of temperature-responsive polymer-protein conjugates has been used to enhance purifications using pNIPAm-modified Protein A and monoclonal antibodies that were synthesized with the “grafting-to” approach. “Grafted-to” enzyme-pNIPAm conjugates are unpredictable however in that some enzymes exhibit modulated bioactivity but others do not.
In order to provide an alternative approach to synthesis of polymer-enzyme conjugates that would allow higher densities, and finer site control, a protein surface initiated “grafting from” technique was previously developed (Lele et al, Biomacromolecules 2005, 6, 3380-3387) This resulted in a higher density of polymer on the enzyme surface but because the initiator binding and polymerization were done in an organic solvent-water biphasic medium the recovery of activity was low and the density was still not optimal. (see also Heredia, K. L.; Bontempo, D.; Ly, T.; Byers, J. T.; Halstenberg, S.; Maynard, H. D. J Am Chem Soc 2005, 127, 16955)
“Grafting from” techniques initiate polymerization directly from the surface of proteins using controlled radical polymerization. Most often, either atom transfer radical polymerization (ATRP) (see Lele, B. S.; Murata, H.; Matyjaszewski, K.; Russell, A. J. Biomacromolecules 2005, 6, 3380; Nicolas, J.; San Miguel, V.; Mantovani, G.; Haddleton, D. M. Chem. Commun. 2006, 4697; Heredia, K. L.; Bontempo, D.; Ly, T.; Byers, J. T.; Halstenberg, S.; Maynard, H. D. J. Am. Chem. Soc. 2005, 127, 16955; Qi, Y.; Amiram, M.; Gao, W.; McCafferty, D. G.; Chilkoti, A. Macromol. Rapid Commun. 2013, 34, 1256; Gao, W.; Liu, W.; Mackay, J. A.; Zalutsky, M. R.; Toone, E. J.; Chilkoti, A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 15231) or reversible-addition fragmentation chain transfer (RAFT) (Liu, J.; Bulmus, V.; Herlambang, D. L.; Barner-Kowollik, C.; Stenzel, M. H.; Davis, T. P. Angew. Chem., Int. Ed. 2007, 46, 3099; De, P.; Li, M.; Gondi, S. R.; Sumerlin, B. S. J. Am. Chem. Soc. 2008, 130, 11288) are used, because each provide low polydispersity indices (PDI), a large library of monomers, and biologically relevant reaction conditions (aqueous solvent and ambient temperature). In “grafting from,” unreacted monomer is easily separated from the bioconjugate and high polymer density is achieved more easily due to the lack of steric limitations seen in “grafting to.” One drawback to “grafting from” is the necessity to have vinyl monomers for radical polymerization. Thus, some polymers, such as PEG, must be slightly modified to use with the “grafting from” approach.
Another major limitation of the current “grafting-from” ATRP techniques for polymer based protein engineering is in the attachment or immobilization of an ATRP-initiator to the enzyme. Until recently most functionalized ATRP initiator compounds were insoluble or of low solubility in aqueous solution. Thus, immobilization of an ATRP initiator to a protein was performed in mixtures of water and organic solvents such as dichloromethane, methanol, DMF, or DMSO. (Nicolas, J.; San Miguel, V.; Mantovani, G.; Haddleton, D. M. Chem. Commun. 2006, 46, 4697-4699; Magnusson, J. P.; Bersani, S.; Salmaso, S.; Alexander, C.; Caliceti, P. Bioconjugate Chem. 2010, 21, 671-678; Yaayan, G.; Saeed, A. O.; Fernández-Trillo, F.; Allen, S.; Davies, M. C.; Jangher, A.; Paul, A.; Thurecht, K. J.; King, S. M.; Schweins, R.; Griffiths, P. C.; Magnusson, J. P.; Alexander, C. Polym. Chem. 2011, 2, 1567-1578; Averick, S.; Simakova, A.; Park, S.; Konkolewicz, D.; Magenau, A. J. D.; Mehl, R. A.; Matyjaszewski, K. ACS Macro Lett. 2011, 1, 6-10.) Such mixtures often lead to inactivation and/or denaturation of enzymes during the immobilization reaction. Further, initiator immobilizations performed on chymotrypsin (CT) in a biphasic solution and on trypsin in 2% DMSO resulted in 21-50% and 46% occupation of available conjugation sites, respectively.
Techniques to synthesize protein-polymer conjugates have developed rapidly in recent years due to advancements in both protein and polymer science. One of the first, and still most common polymers to attach to proteins is poly(ethylene glycol) (PEG), (see Alconcel, S. N. S.; Baas, A. S.; Maynard, H. D. Polym. Chem. 2011, 2, 1442), which imparts stealth properties on the protein by reducing immunogenicity and increases in vivo stability by slowing renal clearance and degradation. However, this polymer does not add specific functionality to the protein and often results in reduced activity. (see Veronese, F. M. Biomaterials 2001, 22, 405) More recently, different polymers have been utilized to synthesize “smart conjugates” (see Hoffman, A. S.; Stayton, P. S. Prog. Polym. Sci. 2007, 32, 922 that respond to external stimuli such as pH (Lackey, C. A.; Murthy, N.; Press, O. W.; Tirrell, D. A.; Hoffman, A. S.; Stayton, P. S. Bioconjugate Chem. 1999, 10, 401; Strozyk, M. S.; Chanana, M.; Pastoriza-Santos, I.; Pérez-Juste, J.; Liz-Marzán, L. M. Adv. Funct. Mater. 2012, 22, 1436.). In addition, specific polymer choices for tailored applications, such as increased substrate affinity (Keefe, A. J.; Jiang, S. Y. Nat. Chem. 2012, 4, 60) have been reported. Polymer-based protein engineering refers to these tailored polymer conjugation applications that target problems that previously could only potentially be solved with molecular biology-dependent techniques.
Poly(sulfobetaine methacrylamide) (pSBAm) and poly(N-isopropylacrylamide) (pNIPAm) are two polymers that have been investigated for a wide range of chemical and biological applications. Specifically, pNIPAm can be used in applications for cardiac repair (Naito, H.; Takewa, Y.; Mizuno, T.; Ohya, S.; Nakayama, Y.; Tatsumi, E.; Kitamura, S.; Takano, H.; Taniguchi, S.; Taenaka, Y. ASAIO J. 2004, 50, 344), protein drug release, and biomolecule separations (Zhou, P.; Yu, S. B.; Liu, Z. H.; Hu, J. M.; Deng, Y. Z. J. Chromatogr. A 2005, 1083, 173). pSBAm is used frequently for non-fouling surface modification (Zhang, Z.; Finlay, J. A.; Wang, L.; Gao, Y.; Callow, J. A.; Callow, M. E.; Jiang, S. Langmuir 2009, 25, 13516; Smith, R. S.; Zhang, Z.; Bouchard, M.; Li, J.; Lapp, H. S.; Brotske, G. R.; Lucchino, D. L.; Weaver, D.; Roth, L. A.; Coury, A.; Biggerstaff, J.; Sukavaneshvar, S.; Langer, R.; Loose, C. Sci. Transl. Med. 2012, 4, 153ra132). Both pSBAm and pNIPAm respond to changes in temperature by predictable alterations in polymer folding. pNIPAm has a lower critical solution temperature (LCST), where above ˜32° C. in deionized water the polymer experiences a reversible collapse, in which it becomes hydrophobic and dehydrated. (Schild, H. G. Prog. Polym. Sci. 1992, 17, 163.) pSBAm exhibits a similar, but opposite behavior known as upper critical solution temperature (UCST) phase transition. pSBAm UCST values are more dependent on molecular weight than the LCST of pNIPAm, but below a given temperature polymer chains collapse from a coil to globule orientation as they phase separate and become insoluble in aqueous media. (Chen, L.; Honma, Y.; Mizutani, T.; Liaw, D. J.; Gong, J. P.; Osada, Y. Polymer 2000, 41, 141.) Free block copolymers with both UCST and LCST properties have been reported previously (Arotcarena, M.; Heise, B.; Ishaya, S.; Laschewsky, A. J. Am. Chem. Soc. 2002, 124, 3787; Weaver, J. V. M.; Armes, S. P.; Butun, V. Chem. Commun. 2002, 2122), but protein-polymer conjugates are most often only synthesized with single temperature responsiveness imparted by homopolymer conjugation (Kulkarni, S.; Schilli, C.; Muller, A. H. E.; Hoffman, A. S.; Stayton, P. S. Bioconjugate Chem. 2004, 15, 747; Boyer, C.; Bulmus, V.; Liu, J. Q.; Davis, T. P.; Stenzel, M. H.; Barner-Kowollik, C. J. Am. Chem. Soc. 2007, 129, 7145). While block copolymers are sometimes conjugated to proteins with the “grafting to” approach, there are few reports of block copolymers being grown from proteins using “grafting from.” Previously, Sumerlin and coworkers used “grafting from” to synthesize a block copolymer using two consecutive RAFT polymerizations from lysozyme (Li, H. M.; Li, M.; Yu, X.; Bapat, A. P.; Sumerlin, B. S. Polym. Chem. 2011, 2, 1531) and bovine serum albumin (Li, M.; Li, H. M.; De, P.; Sumerlin, B. S. Macromol. Rapid Commun. 2011, 32, 354.). Kulkarni et al. synthesized a block copolymer with modified temperature sensitivity, but used the “grafting to” process for protein conjugation (see Kulkarni, S.; Schilli, C.; Grin, B.; Muller, A. H. E.; Hoffman, A. S.; Stayton, P. S. Biomacromolecules 2006, 7, 2736).